Nanoscale Flow Cytometry

"Nanoscale flow cytometry, also called Flow Virometry, is a new and powerful tool in the field of virology that enables the phenotypic analysis of the markers at the surface of individual virions. Virus populations can now be profiled and sorted in multi-parameter analyses, much in the same way as cells"

Scientific Reports vol 7, Article number: 17769 (2017)

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Beckman Coulter MoFlo Astrios EQ

BD SORP LSRFortessa (300mW 488nm)

Beckman Coulter Cytoflex S

Enveloped Viruses as Biological Reference Material

Nanoscale flow cytometry (NFC) is becoming a method of choice for the phenotypic analysis of extracellular vesicles (EVs) and virus particles. Some clinically relevant viruses such as HIV and small EVs are less than 200nm in diameter, which places them at the limit of detection for many commercial flow cytometers. Optimization of both instrument settings as well as preparation of samples for NFC is therefore crucial for the successful analysis of these particles. Although efforts are being made to develop biological reference materials, with physical properties closer to those of the particles of interest, the most common reference material for NFC still remains synthetic beads (polystyrene or silica). These beads are not ideal reference materials since they have higher refractive indices and emit much higher fluorescence than biological particles, and often cannot be labeled with the same dyes as the biological particles of interest.

Enveloped viruses share close structural similarities with EVs in that the virus envelope is derived from cellular membranes. Due to the inherent physical uniformity of virus particles, they are easy to locate by scatter and fluorescence detection. They are naturally occurring in a range of sizes from 50-350nm in diameter. They express distinct and physiologically relevant amounts of molecules such as nucleic acids, phospholipids, and proteins at similar levels to EVs and can potentially be used as experimental controls for EV studies. They can also be genetically inactivated to address biosafety concerns and engineered to express gene products of choice on their surface. For these reasons, enveloped viruses, especially retroviruses, make suitable candidates for use as reference materials for both instrument set-up as well as experimental controls.

CYTO2018 - Abstract #150

Scientific Reports vol 7, Article number: 17769 (2017)

HIV Virus Phenotyping & Discrimination from Extracellular vesicles

To achieve a cure for HIV, all cells harbouring an infectious HIV provirus must be eliminated, or permanently neutralized in absence of cART. Additionally, to complicate matters, there is growing experimental evidence that there is residual viral replication and evolution in infected cell reservoirs in patients on cART. These findings further reinforce the notion that the virus continues to replicate in anatomical sites unreachable by standard antiviral drugs. The way forward for designing a cure for HIV is to first identify the cell types and subsets that constitute the latent infected cell reservoir and also the actively replicating reservoir in patients on cART. This poses a considerable challenge because these cells are extremely rare, dispersed throughout the infected host, and may reside in immune privileged sites.

When latency is reversed in an infected reservoir cell, it produces vast amounts of virus that is released in the blood. HIV-1 is an enveloped virus that buds and is released from the cell surface, taking with it parts of the cellular membrane. Cell surface receptors, lipids and markers are therefore transferred to the surface of the virus. This process is not random, as several abundantly expressed cell surface markers are not detected on HIV-1 virions.

We hypothesize that analyzing, profiling and quantifying host molecules at the surface of individual HIV-1 virions will provide valuable information on the identity of the infected parental cell actively releasing virus and the relative contribution of each reservoir cell subset to the overall viremia